Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Blood. 2000 Jun 15;95(12):3645-51.

Abstract

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, CD / genetics*
  • Antigens, CD / physiology
  • Antigens, CD34 / blood
  • Cell Line
  • Cells, Cultured
  • Fibrin / metabolism
  • Flow Cytometry
  • Genetic Therapy*
  • Humans
  • Integrin beta3
  • Integrins / genetics
  • Megakaryocytes / physiology*
  • Phenotype
  • Platelet Membrane Glycoproteins / genetics*
  • Platelet Membrane Glycoproteins / physiology
  • Polymerase Chain Reaction
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Thrombasthenia / blood
  • Thrombasthenia / genetics*
  • Thrombasthenia / therapy*
  • Transfection

Substances

  • Antigens, CD
  • Antigens, CD34
  • Integrin beta3
  • Integrins
  • Platelet Membrane Glycoproteins
  • Recombinant Proteins
  • Fibrin